Imino proton NMR guides the reprogramming of A•T specific minor groove binders for mixed base pair recognition

Abstract

Sequence-specific binding to DNA is crucial for targeting transcription factor-DNA complexes to modulate gene expression. The heterocyclic diamidine, DB2277, specifically recognizes a single G•C base pair in the minor groove of mixed base pair sequences of the type AAAGTTT. NMR spectroscopy reveals the presence of major and minor species of the bound compound. To understand the principles that determine the binding affinity and orientation in mixed sequences of DNA, over thirty DNA hairpin substrates were examined by NMR and thermal melting. The NMR exchange dynamics between major and minor species shows that the exchange is much faster than compound dissociation determined from biosensor–surface plasmon resonance. Extensive modifications of DNA sequences resulted in a unique DNA sequence with binding site AAGATA that binds DB2277 in a single orientation. A molecular docking result agrees with the model representing rapid flipping of DB2277 between major and minor species. Imino spectral analysis of a 15N-labeled central G clearly shows the crucial role of the exocyclic amino group of G in sequence-specific recognition. Our results suggest that this approach can be expanded to additional modules for recognition of more sequence-specific DNA complexes. This approach provides substantial information about the sequence-specific, highly efficient, dynamic nature of minor groove binding agents.

First Structure of a Designed Minor Groove Binding Heterocyclic Cation that Specifically Recognizes Mixed DNA Base Pair Sequences

“…[14] Similarly, B5 and B6 phenyl protons also show strong intermolecular NOE contacts with the imino proton of G5 in the DNA-DB2277 complex (Table 1). The assignments for the strong intermolecular cross peaks observed between the H2 of adenine and phenyl protons of DB2277 in NOESY spectrum are shown in Figure 7.…”

“…A single and selective binding orientation of the DB2277-DNA complex is observed in imino proton spectra with single resonance peaks for each base pair (Figure 2). [14] 1D proton NMR experiments of the DNA hairpin with 15 N-labeled central G in the –AAGATA– binding site were conducted with DB2277 and reported previously. [14] The findings from this experiment along with 1D NOE experiments conducted on the DB2277-DNA complex allow the assignment of the peak at 11.5 ppm to a downfield shifted aza-benzimidazole-NH (BI -NH) proton signal of DB2277.…”

“…[14] The findings from this experiment along with 1D NOE experiments conducted on the DB2277-DNA complex allow the assignment of the peak at 11.5 ppm to a downfield shifted aza-benzimidazole-NH (BI -NH) proton signal of DB2277. [14] …”

Systematic synthetic and biophysical development of mixed sequence DNA binding agents

“…We recently reported that DB2120 with a central pyridine linker unit flanked by phenyl-benzimidazole-based modules for AT recognition (Figure 1) could strongly and selectively recognize a single G base in a G·C bp flanked by AT sequences with an nM affinity (31). It is an important new design finding and complements other new minor groove binders that can also target mixed sequences of DNA (35, 36). These compounds provide strong evidence that new heterocyclic derivatives, with appropriate positioning of H-bond acceptors to interact with the G amino -NH which projects into the minor groove, can be successfully designed.…”

Lithium Hexamethyldisilazane Transformation of Transiently Protected 4-Aza/Benzimidazole Nitriles to Amidines and their Dimethyl Sulfoxide Mediated Imidazole Ring Formation

“…10,11 Furthermore, rational design of azaBI-based DNA minor groove binders led to morphing traditional BI recognition of DNA from AT specific 12–15 to guanine (G)-containing sequence specific (Figure 1) by introducing the aza group. 16–18 This represents a key milestone showing that small molecules can be structured to recognize any given sequence on DNA. Targeting DNA at specific sequences augments a therapeutic strategy of controlling gene expression as a venue for treating many intractable diseases.…”